Low-molecular weight active ingredient extract from yeasts and method for producing it

ABSTRACT

The invention relates to low-molecular, metabolism-activating mixtures of active ingredients from yeast fungi of the order Saccharomycetes and is characterized in that they are obtained from yeasts of the said order of Saccharomycetes, which are cultivated for some hours at temperatures starting at 37° C., are heated to a maximum of 45° C. and, following cooling, are subsequently processed in a manner known per se.

This is a division of application Ser. No. 08/768,622, filed Dec. 18,1996, which is a continuation of PCT/EP96/01886 filed May 7, 1996.

The invention relates to low-molecular weight active ingredients fromyeasts and to a method for producing same.

Yeast extracts from yeast fungi of the order Saccharomycetes, as well astheir anabolic and respiration-stimulating and generallymetabolism-activating effects, are known. Methods for obtaining suchextracts have been described, for example, in EP-B 0 065 246. In thismethod, after a proteolytic enzyme had been added at the time of optimumfermentation, a suspension of the yeast fungus is subjected to anultrasonic treatment until the temperature had been noticeablyincreased. Although the use of ultrasonics for the dialysis of yeastcells is an effective means for obtaining the extracts, it has beenfound that the ultrasonic treatment has quite negative side effects,since practically all compounds in an aqueous solution are subjected toa more or less pronounced oxidation. Thus, no genuine active agents areextracted from the cells in the course of the treatment withultrasonics, instead, as a rule they are chemically altered compounds.

There is still a demand for yeast extracts and methods for producingthem, which result in as genuine as possible a product and have thelowest possible content of substances altered by the production method.

To attain this object, yeast extracts in accordance with claim 1 areproposed, as well as methods for their production in accordance withclaim 2.

It has been surprisingly found that it is possible to produce yeastextracts with metabolism-activating effects in a qualitatively andquantitatively improved form, if the cultures are subjected in a definedmanner to a temperature change. In accordance with the invention theyeasts, which are maintained at approximately 15° C. prior to preparingthe culture, are suspended in water, wherein the ratio of yeast:water isapproximately 1:2. Subsequently an amount of sugar, in particularsaccharose, which can be fermented with the respective yeast, is admixedin amounts of 5 weight-%. The mass is then heated to the optimumcultivation temperature of 37° to 38° C. and maintained at thistemperature for at least 3 hours, and brief stirring and aerating isperformed at set intervals. This generation time of the yeasts isapproximately 60 minutes.

After three hours of cultivation, the mass then is heated relativelyquickly to maximally 45° C. and maintained at that temperature for 60minutes. Subsequently the suspension is allowed to slowly cool to 25° C.during a period of 60 minutes and is maintained at this temperature forapproximately 60 minutes. Thus the total time of the production processlasts approximately 6 hours.

The temperature resistance of the yeasts is improved by increasing thecultivation temperature to maximally 45° C. during a generation time,and the total metabolism is clearly increased. It is assumed that theincreased yield of low-molecular weight, metabolism-active ingredientsis the result of the increased metabolization during the increasedgrowth temperature. This assumption is supported in that heat shockproteins can be found in the active ingredient concentrate. Heatproteins, hsp for short, are proteins which are briefly synthesized atgreatly increased rates by living cells during thermal or chemicalstress. Their exact function is not yet known, but they are essentialfor surviving stress situations, and it is suspected that they initiateor support the ATP-dependent refolding of denatured proteins. Proteinsof the hsp family 60 and 70 are contained in the yeast hydrolizates.Isolation and proof of hsp are described, for example, in Nature(London) 337, 655 to 659 and 44 to 47 (1989).

Saccharomyces cerevisiae, also in various pure culture forms, orSaccharomyces uvarum or Saccharomyces rosei, are preferably used as theyeasts.

Following the approximately total culture time of 6 hours, the mass iscomminuted with the aid of suitable mechanical mills, such as colloidmills. If necessary, comminution can also be performed following theenzyme addition. Then the mass is reacted at approximately 37° to 38° C.with a proteolytic enzyme or a mixture of such enzymes, wherein theratio of enzyme:biomass should be approximately 0.03:1. The reactiontime is a function of the type of proteolytic enzyme used and as a ruleis approximately 180 minutes. Papain, ficine or bacterial or fungalproteases are preferably employed.

Following solubilization, the entire suspension is heated inside of 30minutes to 85° C. for activating the enzyme, and is maintained at thistemperature for 30 minutes. After cooling, the mass is centrifuged,wherein the sediment can be washed one more time if required. Theresidue containing the active ingredients sought is filtered andconcentrated at temperatures not above 40° C. in vacuo. Subsequentlythis solution can be subjected to usual and known spray-dryinggranulation.

The mixtures of active ingredients in accordance with the inventionpresent clearly elevated metabolic activities in comparison with themixtures of active ingredients known so far and produced in accordancewith other methods, which, in the fibroblast test, as a rule are clearlyhigher, in part by 50 to 100%, than those of the products known up tonow.

The mixtures of active ingredients in accordance with the invention canbe employed in human medicine and veterinary medicine in all those casesin which an activation of the metabolism is necessary, for example forencouraging the healing of slow-healing wounds, for improved utilizationof food, in particular in stock-breeding and pisciculture. A furtherarea of use is in the field of stimulating the activities ofmicroorganisms in enzymatic processes when treating foodstuffs.

The invention will be described in detail below by means of an example:

10 kg of Saccharomyces cerevisiae of the bottom yeast culture type arereacted with 20 kg of purified and filtered water of drinking waterquality in a reactor. This suspension is provided with 5 weight-% ofsaccharose and well mixed by stirring. Then the suspension is heated to37° to 38° C. and maintained at this temperature for at least 3 hours,wherein brief stirring and aeration is performed at intervals ofapproximately half an hour. Following this the mass is heated to 45° C.and maintained for sixty minutes at this temperature, and afterwardsagain cooled to 25° C. over a period of time of 60 minutes andmaintained there for 60 minutes.

Subsequently the balanced suspension is reacted with an appropriateamount of papain, wherein the ratio of enzyme:biomass should be 0.003:1.Following the enzyme addition with simultaneous comminution of theyeasts, the mixture is stirred for 180 minutes at 37° to 38° C. Then themixture is heated to 85° C. within 30 minutes and maintained at thistemperature for 30 minutes, and then cooled and centrifuged. Thecentrifuged material is drawn off and the sediment is washed again, andagain centrifuged. The centrifuged materials are combined and then,following filtration, concentrated at temperatures below 40° C.,preferably with the aid of a vacuum evaporator. The concentratedsolution which is free of cells is then subjected in a manner known perse to spray-drying granulation.

I claim:
 1. A method for producing a mixture of low-molecular weight,metabolically active ingredients comprising heat shock proteins of oneor both of the families hsp 60 and 70, by:(a) cultivating liveSaccharomycetes yeast which contain heat shock proteins of one or bothof the families hsp 60 and 70, in a solution at a temperature between37-38° C., (b) subsequently heating the yeast solution to about 45° C.,(c) slowly cooling the yeast solution to 25° C., and (d) isolating fromthe yeast solution a mixture of low-molecular weight, metabolicallyactive ingredients comprising heat shock proteins of one or both of thefamilies hsp 60 and 70,wherein the yeast remains viable and intactthroughout steps (a), (b) and (c).
 2. The method according to claim 1,wherein in step (a) the yeast solution is cultivated at a temperaturebetween 37-38° C. for 2-6 hours, in step (b) the yeast solution isheated to 45° C. for 40-80 minutes, and in step (c) the yeast solutionis cooled to 25° C. within 40-80 minutes, and is maintained at 25° C.for about 40-80 minutes.
 3. The method according to claim 1 or 2,wherein step (a) further includes adding a fermentable sugar to theyeast solution in an amount of 5%.
 4. The method according to claim 1,wherein in step (a) the yeast is cultivated at a temperature between37-38° C. for 2-6 hours.
 5. The method according to claim 1, wherein theSaccharomycetes yeast is selected from the group consisting ofSaccharomyces cerevisiae, Saccharomyces uvarum and Saccharomyces rosei.6. The method according to claim 2, wherein in step (b) the yeastsolution is heated to 45° C. for 40-80 minutes.
 7. The method accordingto claim 2, wherein in step (c) the yeast solution is cooled to 25° C.for 40-80 minutes.
 8. The method according to claim 1 or 2, wherein step(d) includes comminuting and enzymatically solubilizing the yeast in thepresence of proteolytic enzymes at a temperature of 37°-38° C., heatingthe yeast solution above 80° C. to effect activation of the proteolyticenzymes, centrifuging the yeast solution, and isolating a mixture oflow-molecular weight, metabolically active ingredients and heat shockproteins of one or both of the families hsp 60 and 70 is isolatedtherefrom.
 9. The method according to claim 8, wherein the proteolyticenzyme is papain.
 10. The method according to claim 9, wherein theproteolytic enzyme is papain, ficine, bacterial protease or fungalprotease.
 11. The method according to claim 8, wherein the ratio ofproteolytic enzymes to yeast in step (d) is approximately 0.003:1, byweight.
 12. The method according to claim 8, wherein Saccharomycescerevisiae is the yeast.